The economic impact of infectious diseases in the poultry industry is well-appreciated. Immunization of birds has helped reduce the cost of production by decreasing the incidence of-gastrointestinal, respiratory and systemic diseases. While vaccines provide adequate immunity for those pathogens against which a flock has been immunized, there are few vaccines which can provide broad-based cross-protection against unanticipated diseases or against those diseases for which an animal has not been specifically vaccinated.
A number of important diseases of domestic poultry are caused by bacteria able to invade host tissues, such as Salmonella spp., Escherichia spp. and Pasteurella spp. While many vaccines are available for immunization against individual species and serotypes, none provide cross-protection or stimulate broad-based immunity against multiple serotypes, species or genera.
One essential factor required for a bacteria to induce clinical disease is the ability to proliferate successfully in a host tissue. Iron is an essential nutrient for the growth of gram-negative bacteria in vivo, but is virtually unavailable in mammalian and/or avian tissues because the iron is either intracellular or extracellular, complexed with high affinity, iron-binding proteins, for example, transferring in blood and lymph fluids and lactoferrin in external secretions. In normal tissues, the concentration of iron is approximately 10xe2x88x9213M, far below that required for bacterial growth.
To circumvent these restrictive conditions, pathogenic bacteria have evolved high affinity iron transport systems produced under low iron conditions, which consist of specific ferric iron chelaters, xe2x80x9csiderophores,xe2x80x9d and iron-regulated outer membrane proteins (IROMPs) and/or siderophore receptor proteins (SRPs) which are receptors for siderophores on the outer membrane of the bacterial cell. Siderophores are synthesized by and secreted from the cells of gram-negative bacteria under conditions of low iron. Siderophores are low molecular weight proteins ranging in molecular mass from about 500 to about 1000 MW, which chelate ferric iron and then bind to IROMPs in the outer bacterial membrane which, in turn, transport the iron into the bacterial cell. Although the use of IROMPs as immunogens has been considered, these proteins have not been examined for such use, at least in part, due to an inability to extract these proteins from bacterial membranes in high volume and with a desired level of purity and immunogenic quality.
Accordingly, an object of the invention is to provide a method for obtaining high amounts of immunogenic quality siderophore receptor proteins from Escherichia coli, Salmonella, Pasteurella, and other gram-negative bacteria. Another object is to provide a vaccine for immunizing poultry and other animals against these bacteria. Yet another object is to provide a vaccine for cross-protect-on against multiple serotypes, species and/or genera of bacteria belonging to the family Enterobacteriaceae and/or Pasteurellaceae. A further object is to provide a diagnostic assay to monitor and/or profile sepsis and subclinical disease caused by gram-negative bacteria under field conditions.
These and other objects are achieved by the present invention which is directed to a vaccine for prevention and treatment of infection by gram-negative bacteria, and a method of immunizing poultry and other animals against such infections using the vaccine. The invention also provides a method for isolating and purifying outer membrane siderophore receptor proteins from gram-negative bacteria for producing the vaccine. The invention further provides an in vitro method of diagnosing infections of gram-negative bacteria in an animal using antibodies raised to the isolated receptor proteins.
The vaccine is useful for immunizing an avian or other animal against infection by gram-negative bacteria such as colibacillosis, salmonellosis and pasteurellosis. The vaccine is composed of a substantially pure siderophore receptor protein derived from the outer membrane of a gram-negative bacteria, for example, Salmonella spp., Escherichia spp. and Pasteurella spp. A siderophore receptor protein, useful according to the invention, is a protein or antigenic peptide sequence thereof derived from the outer membrane of a gram negative bacterium, which is capable of producing an antibody that will react with the siderophore receptor protein expressed by a gram-negative bacteria of the same or different strain, species or genus. Preferably, the siderophore receptor protein is derived from a bacterium belonging to the family Enterobacteriaceae and/or Pasteurellaceae.
The vaccine contains siderophore receptor proteins (SRPs) derived from a gram-negative bacteria, capable of eliciting an immune response in an animal with the production of anti-SRP antibodies. These antibodies will react with siderophore receptor proteins of that bacteria, and may also cross-react with siderophore receptor proteins of a different strain, species and/or genera of gram-negative bacteria to provide cross-protection against infection from such other bacteria. Useful siderophore receptor proteins having a molecular weight of about 72-96 kDa, as determined by SDS-PAGE, have been isolated from E. coli, Salmonella spp., Pasteurella spp., Pseudomonas spp., and Klebsiella spp. Preferably, the siderophore receptor proteins (SRPs) are derived from Escherichia coli, Salmonella spp. and/or Pasteurella spp. The antibodies produced from those SRPs will react with SRPs of those bacteria and cross-react with SRPs of a different strain, species and/or genera of bacteria within the family Enterobacteriaceae and/or Pasteurellaceae.
The vaccine contains one or more siderophore receptor proteins extracted from the outer membrane of a single strain or species, or two or more different strains or species of gram-negative bacteria. The amount and type of siderophore receptor protein included in the vaccine is effective to stimulate production of antibodies reactive with a siderophore receptor protein of one, preferably two or more strains, species or genera of gram-negative bacteria. A preferred vaccine is composed of an amount and profile of siderophore receptor-proteins to effectively induce antibodies reactive with a majority, preferably all, of the siderophore receptor proteins of a bacterial population to effectively enhance opsonization and complement-mediated bacterial lysis, and/or block the iron binding capacity of the bacteria. The siderophore receptor protein is combined with a physiolcgically-acceptable carrier, preferably a liquid. The vaccine may further include an adjuvant to enhance the immune response, and other additives as desired, such as preservatives, flavoring agents, buffering agents, and the like.
The present invention also provides a method for isolating high quantities of immunogenically effective siderophore receptor proteins from outer membranes of a single strain or species of gram-negative bacteria such as E. coli, Salmonella and/or Pasteurella. The method includes culturing the organism under conditions of low iron availability, that is, in a culture medium that lacks iron or includes an iron chelating agent. The siderophore receptor proteins are then separated from the bacterial outer membrane and purified by use of the anionic detergent, sodium dodecyl sulfate, preferably under non-reducing conditions.
The siderophore receptor proteins may be utilized to raise polyclonal antibody sera and monoclonal antibodies for use in passive immunization therapies. Such antibodies may also be used in an in vitro method of diagnosing a gram-negative bacterial infection in an animal. The diagnostic method includes contacting a body material potentially infected with a gram-negative bacteria, such as a tissue sample or body fluid, with a labelled antibody raised to a siderophore receptor protein, and detecting the label in the complex formed between the, siderophore receptor protein in the body material and the labelled antibody. The method may also be performed by combining the body sample with the antibody to the siderophore receptor protein, and then contacting the sample with a labelled anti-species antibody reactive with the protein-specific antibody, and then detecting the label.
The siderophore receptor proteins can also be used as capture antigens in a method of monitoring and profiling gram negative sepsis. For example, the protein may be used in an ELISA technique in which the protein is bound to a solid support and contacted with a sample material to react with and detect antibodies present in the sample.